Investigating Srsf3-mediated alternative RNA splicing in craniofacial development

研究颅面发育中 Srsf3 介导的替代 RNA 剪接

• 批准号: 10533544
• 负责人: Thomas E Forman
• 金额: $ 3.88万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-07-08 至 2025-07-07
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10533544
• 关键词: 1-Phosphatidylinositol 3-Kinase; Ablation; Affect; Alleles; Alternative Splicing; Apoptosis; Arginine; Cell Lineage; Cell Nucleus; Cell membrane; Cells; Cephalic; Cleft Palate; Complex; Congenital Abnormality; Consensus; Consensus Sequence; Craniofacial Abnormalities; Data; Defect; Disease; Ectoderm; Embryo; Event; Face; Feedback; Gene Expression; Gene Expression Regulation; Genes; Goals; Head; Histologic; Human; Immunoprecipitation; In Vitro; Knock-in; Knock-in Mouse; Label; Lead; Ligands; Maps; Mass Spectrum Analysis; Mediating; Membrane; Mesenchyme; Molecular; Morphology; Mus; Mutation; Neural Crest; Neural Crest Cell; Nuclear Proteins; Ontology; PDGFRA gene; PDGFRB gene; Phenotype; Phosphopeptides; Phosphorylation; Phosphotransferases; Physiological; Platelet-Derived Growth Factor; Platelet-Derived Growth Factor alpha Receptor; Play; Post-Translational Protein Processing; Pregnancy; Process; Protein-Serine-Threonine Kinases; Proteins; RNA; RNA Binding; RNA Splicing; RNA-Binding Proteins; Research; Role; Serine; Signal Transduction; Site; Skeletal Development; Small Interfering RNA; Specificity; Stimulus; Structure; Techniques; Testing; Tissues; Transcript; Western Blotting; cleft lip and palate; craniofacial; craniofacial development; craniofacial structure; crosslink; experimental study; in vitro testing; in vivo; innovation; insight; interest; knock-down; mouse model; mutant mouse model; novel; novel therapeutics; nucleocytoplasmic transport; orofacial cleft; phosphoproteomics; recruit; response; stem; transcriptome sequencing

Project Summary Craniofacial development involves complex signaling to coordinate tissue organization to form the head and face, and disruptions in this process result in common congenital malformations. A key question in this field is how external stimuli lead to gene expression changes required to form fully developed craniofacial structures. Signaling through the Platelet-derived growth factor receptor alpha (PDGFRα) plays a critical role in this process, as mutations in PDGFRΑ are associated with cleft lip/palate in humans. Relatedly, Pdgfra mutant mouse models develop a range of phenotypes from cleft palate to complete facial clefting. Phosphatidylinositol 3-kinase (PI3K) is the primary effector of PDGFRα signaling during skeletal development in the mouse, leading to the activation of the kinase Akt. A previous phosphoproteomic screen demonstrated that Akt phosphorylates the RNA-binding protein (RBP) Serine/arginine-rich splicing factor 3 (Srsf3) downstream of PI3K-mediated PDGFRα signaling in mouse embryonic palatal mesenchyme (MEPM) cells, leading to translocation of phosphorylated Srsf3 into the nucleus. Srsf3 is ubiquitously expressed with enhanced expression in the neural crest-derived mesenchyme and overlying ectoderm of mouse facial processes at mid-gestation. Additionally, ablation of Srsf3 in the murine neural crest cell lineage (cKO) results in a severe midline facial clefting phenotype due to defects in proliferation and survival of cranial neural crest cells. Further, RNA-sequencing of Srsf3 cKO facial process mesenchyme identified alternative RNA splicing events that were enriched for transcripts encoding protein serine/threonine kinases, suggesting that alternative splicing may serve as a novel feedback mechanism for intracellular kinase signaling. The goal of this proposal is to test the hypothesis that PI3K/Akt-mediated PDGFRα signaling regulates Srsf3 protein and RNA interactions to affect the alternative RNA splicing of transcripts necessary for craniofacial development. First, Srsf3 will be immunoprecipitated from MEPM cells in the absence or presence of PDGF-AA ligand and analyzed by mass spectrometry to comprehensively map phosphorylation changes in response to PDGFRα signaling. Further, craniofacial phenotypes will be analyzed in a Srsf3 phosphomutant knock-in mouse model to determine the role of Akt- mediated phosphorylation of Srsf3 in craniofacial development. Next, BioID2 proximity labeling and mass spectrometry will be used to identify Srsf3 protein interacting partners in response to PDGFRα signaling in MEPM cells. Finally, Srsf3-RNA interactions will be purified and sequenced in response to PDGFRα signaling in MEPM cells through enhanced crosslinking and immunoprecipitation analysis to identify direct targets of Srsf3 and determine if RNA binding and/or sequence specificity changes upon Srsf3 phosphorylation. This project will determine the molecular mechanisms by which Srsf3 activity is controlled in response to PDGFRa signaling in the facial mesenchyme, thus providing considerable insight into mechanisms underlying gene expression regulation during mammalian craniofacial development.

项目概要 颅面发育涉及复杂的信号传导以协调组织组织形成头部 和面部，这个过程的中断会导致常见的先天畸形。这其中的一个关键问题 领域是外部刺激如何导致形成完全发育的颅面所需的基因表达变化 结构。通过血小板衍生生长因子受体 α (PDGFRα) 发出的信号在 这个过程，因为 PDGFRA 的突变与人类唇裂/腭裂有关。相关地，Pdgfra突变体 小鼠模型形成了从腭裂到完全面裂的一系列表型。磷脂酰肌醇 3-激酶 (PI3K) 是小鼠骨骼发育过程中 PDGFRα 信号传导的主要效应器，导致 参与激酶 Akt 的激活。之前的磷酸化蛋白质组筛选表明 Akt 磷酸化 PI3K 介导的 RNA 结合蛋白 (RBP) 富含丝氨酸/精氨酸的剪接因子 3 (Srsf3) 下游 小鼠胚胎腭间充质 (MEPM) 细胞中的 PDGFRα 信号传导，导致 磷酸化 Srsf3 进入细胞核。 Srsf3 普遍表达，且在神经细胞中表达增强 妊娠中期小鼠面部突起的嵴衍生间充质和上覆外胚层。此外， 小鼠神经嵴细胞谱系 (cKO) 中 Srsf3 的消融会导致严重的中线面部裂 由于颅神经嵴细胞增殖和存活缺陷而导致的表型。此外，RNA测序 Srsf3 cKO 面部过程间充质鉴定出替代 RNA 剪接事件，这些事件被富集 编码蛋白丝氨酸/苏氨酸激酶的转录本，表明选择性剪接可能作为一种新的方法 细胞内激酶信号传导的反馈机制。该提案的目的是检验以下假设： PI3K/Akt 介导的 PDGFRα 信号传导调节 Srsf3 蛋白和 RNA 相互作用以影响替代方案 颅面发育所需的转录本的 RNA 剪接。首先，Srsf3 将从 在不存在或存在 PDGF-AA 配体的情况下对 MEPM 细胞进行质谱分析 全面绘制响应 PDGFRα 信号传导的磷酸化变化。此外，颅面 将在 Srsf3 磷酸突变敲入小鼠模型中分析表型，以确定 Akt-的作用 颅面发育中 Srsf3 介导的磷酸化。接下来，BioID2 邻近标记和质量 光谱测定法将用于识别 Srsf3 蛋白相互作用伙伴，以响应 PDGFRα 信号传导 MEPM 细胞。最后，Srsf3-RNA 相互作用将被纯化并测序以响应 PDGFRα 信号传导 通过增强交联和免疫沉淀分析在 MEPM 细胞中识别 Srsf3 并确定 RNA 结合和/或序列特异性是否因 Srsf3 磷酸化而改变。这 项目将确定响应 PDGFRa 控制 Srsf3 活性的分子机制 面部间质中的信号传导，从而提供对基因潜在机制的深入了解 哺乳动物颅面发育过程中的表达调控。